U.S. patent number 9,449,377 [Application Number 14/049,096] was granted by the patent office on 2016-09-20 for imaging methods and computer-readable media.
This patent grant is currently assigned to The United States of America as Represented by the Secretary of the Department of Health and Human Services. The grantee listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Service, The United States of America, as represented by the Secretary, Department of Health and Human Service. Invention is credited to Ambika Bumb, Keir Cajal Neuman, Susanta Kumar Sarkar.
United States Patent |
9,449,377 |
Sarkar , et al. |
September 20, 2016 |
Imaging methods and computer-readable media
Abstract
One aspect of the invention provides an imaging method
including: (a) acquiring a first fluorescent image of an object of
interest impregnated with fluorescent nanodiamonds; (b) applying a
magnetic field to the fluorescent nanodiamonds in order to decrease
fluorescence of the fluorescent nanodiamonds; (c) acquiring a
second fluorescent image of the object of interest; and (d)
subtracting the second fluorescent image from the first fluorescent
image to produce a resulting image. Another aspect of the invention
provides an imaging method including: (a) applying a time-varying
magnetic field to an object of interest impregnated with
fluorescent nanodiamonds to modulate the fluorescence of the
fluorescent nanodiamonds; (b) acquiring a plurality of fluorescent
images of the object of interest; and (c) for each corresponding
pixel in the plurality of fluorescent images, calculating a
fluorescence intensity using a lock-in technique.
Inventors: |
Sarkar; Susanta Kumar
(Rockville, MD), Bumb; Ambika (Greer, SC), Neuman; Keir
Cajal (Bethesda, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Service |
Bethesda |
MD |
US |
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Assignee: |
The United States of America as
Represented by the Secretary of the Department of Health and Human
Services (Washington, DC)
|
Family
ID: |
50432699 |
Appl.
No.: |
14/049,096 |
Filed: |
October 8, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140099007 A1 |
Apr 10, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61711702 |
Oct 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G06T
5/003 (20130101); G01N 21/6428 (20130101); G01N
21/6486 (20130101); G06K 9/00134 (20130101); G01N
21/6458 (20130101); G06T 7/0012 (20130101); G06T
5/50 (20130101); G01N 2021/6439 (20130101); G06T
2207/30024 (20130101); G06T 2207/10016 (20130101); G06T
2207/20224 (20130101); G06T 2207/20182 (20130101); G06T
2207/20216 (20130101); G06T 2207/10056 (20130101); G06T
2207/10064 (20130101) |
Current International
Class: |
G06K
9/00 (20060101); G06T 5/50 (20060101); G01N
21/64 (20060101); G06T 5/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Fu "Characterization and application of single fluorescent
nanodiamonds as cellular biomarkers", PNAS Jan. 2007, pp. 727-732.
cited by examiner .
Chapman, et al., "Background-free imaging of luminescent
nanodiamonds using external magnetic field for contrast
enhancement," Optics Letters, vol. 38, No. 11, pp. 1847-1849
(2013). cited by applicant .
Igarashi, et al., "Real-Time Background-Free Selective Imaging of
Fluorescent Nanodiamonds in Vivo," Nano Letters, vol. 12(11), pp.
5726-5732 (2012). cited by applicant.
|
Primary Examiner: Yang; Weiwen
Attorney, Agent or Firm: Kilpatrick Townsend & Stockton
LLP
Government Interests
STATEMENT REGARDING GOVERNMENTAL SUPPORT
The present subject matter was made with U.S. government support.
The U.S. government has certain rights in this subject matter. This
work was supported through grant number HL006087-02 BBC from the
National Heart, Lung, and Blood Institute, National Institutes of
Health.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This applications claims priority under 35 U.S.C. .sctn.119(e) to
U.S. Provisional Patent Application Ser. No. 61/711,702, filed Oct.
9, 2012. The entire content of this application is hereby
incorporated by reference herein.
Claims
The invention claimed is:
1. An imaging method comprising: applying energy to an object,
wherein the object is a biological target selected from the group
consisting of a cell, a tissue, an organ and an organism, wherein
the biological target comprises fluorescent nanodiamonds, and
wherein the applied energy consists of optical light and a magnetic
field; determining the fluorescence intensity of the object; and
generating an image of the object from the fluorescence
intensity.
2. The method of claim 1, wherein applying the magnetic field
comprises applying the magnetic field to decrease the fluorescence
of the fluorescent nanodiamonds.
3. The method of claim 2, wherein determining the fluorescence
intensity comprises: acquiring a first fluorescence image of the
object; acquiring a second fluorescence image while applying the
magnetic field; and comparing the second fluorescence image to the
first fluorescence image to produce a resulting image of the
object.
4. The method of claim 3, wherein the image comprises a plurality
of pixels and the comparing is performed on a pixel-by-pixel
basis.
5. The method of claim 3, further comprising repeating the steps a
plurality of times and averaging the resulting images.
6. The method of claim 5, wherein the plurality of times is greater
than 10.
7. The method of claim 3, wherein acquiring the first fluorescence
image and the second fluorescence image comprises applying the
optical light to the object to cause the fluorescent nanodiamonds
to fluoresce.
8. The method of claim 1, wherein applying the magnetic field
comprises applying a time-varying magnetic field to modulate the
fluorescence of the fluorescent nanodiamonds.
9. The method of claim 8, wherein determining the fluorescence
intensity comprises: acquiring a plurality of fluorescence images
of the object, wherein each of the plurality of fluorescence images
comprises a plurality of pixels; and determining the fluorescence
intensity of each of the plurality of pixels using a lock-in
technique.
10. The method of claim 9, wherein acquiring the plurality of
fluorescence images of the object comprises applying the optical
light to the object to cause the fluorescent nanodiamonds to
fluoresce.
11. The method of claim 9, wherein acquiring a plurality of
fluorescent images comprises using a wide-field camera, a confocal
microscope, or a combination thereof.
12. A non-transitory computer readable medium containing program
instructions executable by a processor for performing the method of
claim 1.
Description
BACKGROUND
Autofluorescence from naturally-occurring fluorescent biomolecules
and fixative agents make it difficult to separate useful
fluorescence from unwanted fluorescence due to overlapping emission
spectra. Therefore, autofluorescence limits the capabilities of
tissue and animal imaging. Even sophisticated spectral unmixing
techniques cannot always reliably and accurately separate useful
signal from background fluorescence.
SUMMARY OF THE PRESENT SUBJECT MATTER
One aspect of the invention provides an imaging method including:
(a) acquiring a first fluorescent image of an object of interest
impregnated with fluorescent nanodiamonds; (b) applying a magnetic
field to the fluorescent nanodiamonds in order to decrease
fluorescence of the fluorescent nanodiamonds; (c) acquiring a
second fluorescent image of the object of interest; and (d)
subtracting the second fluorescent image from the first fluorescent
image to produce a resulting image.
This aspect can have a variety of embodiments. The object of
interest can be a biological target. The biological target can be
selected from the group consisting of: a cell, a plurality of
cells, a tissue, an organ, and an organism.
The magnetic field can be generated by a permanent magnet. The
magnetic field can be generated by an electromagnet.
The second fluorescent image can be acquired during application of
the magnetic field.
Step (d) can be performed on a pixel-by-pixel basis.
The method can further include an additional step (e) of repeating
steps (a)-(d) a plurality of times and averaging the resulting
images. The plurality of times can be greater than 10.
Steps (a) and (c) can include applying an absorption wavelength to
the object of interest.
Another aspect of the invention provides a non-transitory computer
readable medium containing program instructions executable by a
processor. The computer readable medium includes: (a) program
instructions that acquire a first fluorescent image of an object of
interest impregnated with fluorescent nanodiamonds; (b) program
instructions that apply a magnetic field to the fluorescent
nanodiamonds in order to decrease fluorescence of the fluorescent
nanodiamonds; (c) program instructions that acquire a second
fluorescent image of the object of interest; and (d) program
instructions that subtract the second fluorescent image from the
first fluorescent image to produce a resulting image.
This aspect can have a variety of embodiments. The second
fluorescent image can be acquired during application of the
magnetic field.
Another aspect of the invention provides an imaging method
including: (a) applying a time-varying magnetic field to an object
of interest impregnated with fluorescent nanodiamonds to modulate
the fluorescence of the fluorescent nanodiamonds; (b) acquiring a
plurality of fluorescent images of the object of interest; and (c)
for each corresponding pixel in the plurality of fluorescent
images, calculating a fluorescence intensity using a lock-in
technique.
This aspect can have a variety of embodiments. The object of
interest can be a biological target. The biological target can be
selected from the group consisting of: a cell, a plurality of
cells, a tissue, an organ, and an organism.
The magnetic field can be generated by a permanent magnet. The
magnetic field can be generated by an electromagnet.
Step (b) can include applying an absorption wavelength to the
object of interest. The plurality of fluorescent images can be
acquired by a wide-field camera. The plurality of fluorescent
images can be acquired by a confocal microscope.
Another aspect of the invention provides a non-transitory computer
readable medium containing program instructions executable by a
processor. The computer readable medium includes: (a) program
instructions that apply a time-varying magnetic field to an object
of interest impregnated with fluorescent nanodiamonds to modulate
the fluorescence of the fluorescent nanodiamonds; (b) program
instructions that acquire a plurality of fluorescent images of the
object of interest; and (c) program instructions that, for each
corresponding pixel in the plurality of fluorescent images,
calculate a fluorescence intensity using a lock-in technique.
Another aspect of the invention provides an imaging method
including: (a) applying an absorption wavelength to an object of
interest impregnated with fluorescent nanodiamonds; and (b) after a
delay of at least at least about 6 nanoseconds, acquiring a
fluorescent image of the object of interest.
This aspect can have a variety of embodiments. The object of
interest can be a biological target. The biological target can be
selected from the group consisting of: a cell, a plurality of
cells, a tissue, an organ, and an organism.
The delay can be greater than about 10 nanoseconds. The delay can
be greater than between about 10 nanoseconds and about 20
nanoseconds.
The absorption wavelength can be generated by a pulsed laser. The
absorption wavelength can be between about 450 nm and about 650 nm
or between about 900 nm and about 1300 nm. The absorption
wavelength can be between about 450 nm and about 650 nm or between
about 850 nm and about 1350 nm.
Another aspect of the invention provides an imaging method
including: (a) applying an absorption wavelength to an object of
interest impregnated with fluorescent nanodiamonds; and (b) after a
delay of at least at least about 4 nanoseconds, acquiring a
fluorescent image of the object of interest.
This aspect can have a variety of embodiments. The object of
interest can be a biological target. The biological target can be
selected from the group consisting of: a cell, a plurality of
cells, a tissue, an organ, and an organism.
The delay can be greater than about 10 nanoseconds. The delay can
be greater than between about 10 nanoseconds and about 20
nanoseconds.
The absorption wavelength can be generated by a pulsed laser. The
absorption wavelength can be between about 450 nm and about 650 nm
or between about 900 nm and about 1300 nm. The absorption
wavelength can be between about 450 nm and about 650 nm or between
about 850 nm and about 1350 nm.
Another aspect of the invention provides a non-transitory computer
readable medium containing program instructions executable by a
processor. The computer readable medium includes: (a) program
instructions that apply an absorption wavelength to an object of
interest impregnated with fluorescent nanodiamonds; and (b) program
instructions that, after a delay of at least at least about 6
nanoseconds, acquire a fluorescent image of the object of
interest.
BRIEF DESCRIPTION OF THE DRAWINGS
For a fuller understanding of the nature and desired objects of the
present invention, reference is made to the following detailed
description taken in conjunction with the accompanying drawing
figures wherein like reference characters denote corresponding
parts throughout the following views.
FIG. 1 depicts the absorption (excitation) and emission spectrums
of diamond nitrogen-vacancy centers.
FIG. 2 depicts an imaging method according to an embodiment of the
present subject matter.
FIG. 3 depicts an example of pixel-by-pixel subtraction of a second
fluorescent image from a first fluorescent image according to an
embodiment of present subject matter.
FIG. 4 depicts an imaging method according to another embodiment of
the present subject matter.
FIG. 5 depicts an imaging method according to another embodiment of
the present subject matter.
FIG. 6 depicts an imaging system according to an embodiment of the
present subject matter.
FIG. 7A depicts the energy level diagram of negatively-charged
nitrogen vacancy (NV) centers. FIG. 7B depicts a field of view
containing FNDs. FIG. 7C depicts the intensity modulation of the
FND depicted in FIG. 7A upon application of a modulating magnetic
field with 0.1 Hz frequency and 100 Gauss amplitude.
FIG. 8A is a scanning confocal image of FNDs on the surface of a
slide. FIG. 8B is a scanning confocal image taken under identical
imaging conditions of the same field of view after the addition of
.about.1 .mu.M ALEXA FLUOR.RTM. 647 dye, which has a comparable
emission spectrum to that of the FNDs. The fluorescence from the
high concentration of ALEXA FLUOR.RTM. 647 dye completely obscures
the fluorescence of the FNDs. FIG. 8C is a background-free image of
the same field of view after processing images in the presence of
the high ALEXA FLUOR.RTM. 647 dye background (as in FIG. 8B). The
difference between pairs of images collected with and without the
magnetic field was computed and 1000 of these difference images
were averaged together to generate the processed image. Through
this processing, images of the diamonds shown in FIG. 8A are
recovered from images with high background like FIG. 8B.
FIG. 9A is a frame of a movie taken by a scanning confocal
microscope. FIG. 9B shows the wide field background-free image
after processing the movie pixel-by-pixel using a lock-in
algorithm. FIG. 9C depicts the application of the lock-in algorithm
to a bright pixel (horizontal coordinate, x=109 and vertical
coordinate, y=111 from the top left corner of the image in FIG. 9A)
corresponding to a FND. FIG. 9D illustrates the same lock-in
algorithm applied to a dark pixel (horizontal coordinate, x=137 and
vertical coordinate, y=107 from the top left corner of the image in
FIG. 9A) corresponding to the background.
FIG. 10A depicts the fluorescence image acquired with conventional
spectral unmixing techniques of the forequarters of a mouse after
injection of FNDs. The image is an overlay of the background
channel (top left) and the FND channel (top right). The site of
injection of the FNDs is visible in the composite but the lymph
node cannot be distinguished. FIG. 10B shows the same mouse imaged
with the pairwise image subtraction technique. The lymph node is
clearly visible in the processed image (top right) that was
produced by pairwise subtraction of fluorescence images (top left)
with the magnetic field on and off. FIG. 10C depicts the same
images as FIG. 10B obtained with the wide-field lock-in technique.
FIG. 10D depicts an image of the opened mouse chest cavity obtained
with pairwise subtraction background free detection. The lymph node
and the point of initial injection (white arrows) are clearly
visible. FIG. 10E depicts the same image as FIG. 10D obtained with
wide-field lock in background free detection. FIG. 10F depicts the
intensity as a function of time for points in the images where
there are FNDs (1 and 3) and two background spots (2 and 4). The
location of the four points are indicated on the images in FIGS.
10B and 10D.
FIG. 11A is an image of a lymph node (LN) injected with
silica-coated FNDs without applied magnetic field. FIG. 11B is an
image of the same field of view during application of a magnetic
field of .about.100 Gauss. FIG. 11C depicts the sum of 20
subtracted images of FIG. 11B from FIG. 11A. The FNDs are observed
as bright spots in the otherwise dark field. FIGS. 11D and 11E
depict the sums of 20 such subtractions when magnetic field was
always OFF and ON, respectively.
FIG. 12A is a two-photon FLIM image of a different region of the
same LN used in FIGS. 11A-11E. Longer lifetimes (indicated by red
color) are believed to be due to FNDs, for which the reported
lifetimes range from .about.10-20 ns. FIG. 12B is a background-free
image of the same field of view obtained by subtracting images
without and with a magnetic field, and adding 10 such
subtractions.
DEFINITIONS
The present subject matter is most clearly understood with
reference to the following definitions:
As used herein, the singular form "a," "an," and "the" include
plural references unless the context clearly dictates
otherwise.
Unless specifically stated or obvious from context, as used herein,
the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein are modified by the term about.
As used herein, the term "background-free image" refers to an image
having a sufficiently high signal-to-noise ratio (SNR) such that
the fluorescence of fluorescent nanodiamonds can be distinguished
from the fluorescence of background elements such as endogenous
proteins. Suitable SNRs include those greater than about 1:1, about
1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about
7:1, about 8:1, about 9:1, and about 10:1.
As used herein, the terms "comprises," "comprising," "containing,"
"having," and the like can have the meaning ascribed to them in
U.S. patent law and can mean "includes," "including," and the
like.
As used herein, the term "fluorescent image" refers to an image
representing one or more fluorescent emissions. For example, the
fluorescent emissions can be between about 625 nm and about 825 nm.
Fluorescent images are typically obtained typically obtained by
applying an absorption wavelength to an object of interest and
simultaneously or after a delay, capturing an image of fluorescent
emissions. Fluorescent images can be obtained using a variety of
devices including fluorescent microscopes. The fluorescent image
can, in some embodiments, be a two-dimensional image consisting of
a plurality of pixels, each of which can be a numerical
representation of the intensity of fluorescence as at particular
location.
As used herein, the term "fluorescent nanodiamond" (abbreviated as
"FND") refers to nanodiamonds that exhibit fluorescence when
exposed to an appropriate absorption (excitation) spectrum. This
fluorescence can be caused by the presence of nitrogen-vacancy (NV)
centers, where a nitrogen atom is located next to a vacancy in the
nanodiamond. The absorption (excitation) and emission spectrums of
silica-coated FNDs having a diameter of about 100 nm are depicted
in FIG. 1. The absorption (excitation) spectrum generally lies
between about 450 nm and about 600 nm with an excitation peak at
about 565 nm. (FNDs can also be excited by a two-photon process in
the wavelength region between about 900 nm and about 1300 nm.) The
emission spectrum generally lies between about 625 nm and about 750
nm, with an emission peak at about 700 nm. (In the working examples
described herein, the samples were excited at 575 nm and a PTI
fluorometer was used to obtain the excitation spectra.)
As used herein, the term "nanodiamond" refers to diamonds having a
largest dimension of less than about 100 nm. For example, the
largest dimension of a nanodiamond can be less than: about 100 nm,
about 90 nm, about 80 nm, about 70 nm, about 60 nm, about 50 nm,
about 40 nm, about 30 nm, about 20 nm, about 20 nm, about 10 nm,
and the like.
As used herein, the term "object of interest" refers to any object
of which a fluorescent image is desired. An object of interest can
be a biological target, for example, a living organism or a sample
of an organism. The object of interest can be an organism, one or
more organs, one or more tissues, and/or one or more cells.
Although the examples described herein are largely directed toward
biological imaging, FNDs and the methods described herein can be
used to increase the single to noise in any imaging application in
which a modulated magnetic field can be applied to the sample. For
example, FNDs and the methods described herein can be used to image
and study flow or morphology in a high background environment.
Unless specifically stated or obvious from context, the term "or,"
as used herein, is understood to be inclusive.
By "specifically binds" is meant recognition and binding to a
target (e.g., polypeptide, cell, surface antigen, and the like),
but which does not substantially recognize and bind other molecules
in a sample, for example, a biological sample.
The term "subject" as used herein, refers to any organism that is
suitable for being imaged by the methods described herein. Such
organisms include, but are not limited to, human, dog, cat, horse,
cow, sheep, goat, mouse, rat, guinea pig, monkey, avian, reptiles,
bacteria, fungi, viruses, and the like.
The term "tissue" as used herein, refers to a subject's body.
Nonlimiting examples of tissues include tissues from organs such as
brain, heart, lung, liver, stomach, pancreas, colon, rectum,
intestines, blood vessels, arteries, and the like.
Ranges provided herein are understood to be shorthand for all of
the values within the range. For example, a range of 1 to 50 is
understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the
context clearly dictates otherwise).
DETAILED DESCRIPTION OF THE PRESENT SUBJECT MATTER
Nitrogen vacancy centers in nanodiamonds are unique fluorescent
sources that do not photobleach or blink. Remarkably, the
fluorescence intensity of these fluorescent nanodiamonds can be
modulated by a magnetic field of moderate strength (.about.0.01 T).
Furthermore the fluorescence lifetime of nanodiamonds (.about.10-20
ns) is longer than the lifetime (.ltoreq.5-6 ns) of most
fluorophores that contribute to autofluorescence. Both of these
properties of nanodiamonds can be used to achieve background-free
imaging. The modulation of the fluorescence intensity with applied
magnetic field is a unique feature of the fluorescent nanodiamonds,
which in combination with their other features enables a number of
novel imaging applications. Background-free imaging is one of these
applications.
The discovery allows background-free imaging of fluorescent
nanodiamonds in tissue samples and in vivo, where conventional
imaging is difficult due to background fluorescence. We present
several techniques to reduce or eliminate background florescence by
exploiting properties of the fluorescent nanodiamonds. In
particular, magnetic field modulation of the fluorescence intensity
offers a simple, robust, and easily adaptable method to obtain
background-free imaging in a variety of imaging modalities, i.e.,
fluorescence microscopy, confocal fluorescence microscopy, and
wide-field fluorescence animal imaging.
In one embodiment of the present subject matter, subtracting an
image acquired with the magnetic field from one without the field
collected under otherwise identical conditions eliminates constant
background fluorescence while highlighting the diamond fluorescence
that is specifically reduced in one image.
In another embodiment, the field is modulated sinusoidally while
images are acquired. Phase-sensitive detection of the modulated
intensity can then be achieved by post-processing for camera-based
imaging or through lock-in techniques in confocal-based imaging.
This technique could be adapted for use in wide-field and confocal
imaging systems. Importantly, this technique makes use of
conventional continuous wave illumination.
Yet another embodiment makes use of the long excited state lifetime
of the fluorescent nanodiamonds to reject shorter-lived background
fluorescence. This technique relies on a pulsed laser and
time-gated or lifetime imaging. Fluorescent nanodiamonds can be
imaged with two-photon approaches, facilitating these
lifetime-based background rejection techniques.
Methods of Producing Background-Free Image by Subtraction
Referring now to FIG. 2, an imaging method 200 is provided.
In step S202, a first fluorescent image is acquired of an object of
interest impregnated with fluorescent nanodiamonds. The fluorescent
image can be acquired using conventional fluorescent imaging
devices as described herein.
In step S204, a magnetic field is applied to the fluorescent
nanodiamonds in order to decrease fluorescence of the fluorescent
nanodiamonds. The magnetic field can be applied, for example, with
a permanent magnet or an electromagnet. The magnetic field
modulates the fluorescence intensity of the fluorescent
nanodiamonds such that the fluorescence intensity of the
fluorescent nanodiamonds is less than in the first fluorescent
image.
In step S206, a second fluorescent image is acquired of the object
of interest. Preferably, the second fluorescent image has identical
parameters to the first fluorescent image other than the decreased
fluorescence intensity of fluorescent nanodiamonds as a result of
the application of the magnetic field. The second fluorescent image
is acquired while the magnetic field is applied.
In step S208, the second fluorescent image is subtracted from the
first fluorescent image to produce a resulting image. The
subtraction can be performed on a pixel-by-pixel basis. For
example, as shown conceptually in FIG. 3, a second 10.times.10
pixel fluorescent image can be subtracted from a first 10.times.10
pixel fluorescent image to produce a resulting image. Because the
only difference between the first fluorescent image and the second
fluorescent image is the modulation of fluorescent nanodiamonds,
the background fluorescence (e.g., from endogenous proteins) will
be canceled out by the subtraction to produce a background-free
fluorescent image.
Pixel-by-pixel subtraction can be performed manually or can be
automated. A variety of commercially-available computer programs
can perform image subtraction including, for example, MATLAB.RTM.
software available from The MathWorks, Inc. of Natick, Mass. and
IMAGEJ software available from the National Institutes of Health at
http://rsbweb.nih.gov/ij/.
In step S210, steps S202-S208 are repeated a plurality of times
(e.g., greater than 10) and the resulting images are averaged on a
pixel-by-pixel basis to improve the imaging quality.
Methods of Producing Background-Free Images Using Lock-In
Techniques
Referring now to FIG. 4, another imaging method 400 is
provided.
In step S402, time-varying magnetic field is applied to an object
of interest impregnated with fluorescent nanodiamonds to modulate
the fluorescence of the fluorescent nanodiamonds. The time-varying
magnetic field can vary cyclically. For example, the magnitude of
the magnetic field can vary sinusoidally. The magnetic field can be
varied by modulating the current applied to an electromagnet or by
modulating the distance between the magnet (either a permanent
magnet or an electromagnet) and the object of interest.
In step S404, a plurality of fluorescent images of the object of
interest are acquired. Preferably, the plurality of fluorescent
images are captured at a plurality of points within a cycle. The
sampling can occur at regular or irregular intervals and can, but
need not, be matched to the frequency of the phase-modified
magnetic field. The plurality of fluorescent images can be acquired
pixel-by-pixel using a point imager such as a confocal microscope
or multiple pixels at a time using a wide-field imager.
In step S406, the fluorescence intensity of each pixel is
calculated using a lock-in technique. Lock-in techniques multiply
the fluorescence intensity in each pixel of the plurality of
fluorescent images by the amplitude of the magnetic field at the
time of each respective fluorescent image and then calculate the
intensity of the appropriately filtered or processed resulting
products. All background fluorescence will not fluctuate in phase
with the magnetic field modulation over all images and will
therefore average to zero. The fluorescence intensity of the
fluorescent nanodiamonds will vary in phase with the magnetic
intensity and, therefore, average to half of the amplitude of the
magnetic intensity.
Lock-in techniques are described in publications such as Richard
Burdett, "Amplitude Modulated Signals--The Lock-in Amplifier," in
Handbook of Measuring System Design (2005) and Stanford Research
Systems, Inc., "About Lock-In Amplifiers: Application Note #3,"
available at
http://www.thinksrs.com/downloads/PDFs/ApplicationNotes/AboutLIAs.pdf.
Generally speaking, single-pixel inputs obtained from point
detectors can processed directly by conventional lock-in amplifiers
available from suppliers such as Stanford Research Systems, Inc. of
Sunnyvale, Calif., while algorithms can be written using software
such as MATLAB.RTM. to step through each set of corresponding
pixels in a series of fluorescent images and perform a lock-in
technique to produce a resulting background-free image.
If the fluorescent images are acquired pixel-by-pixel, a lock-in
technique can be applied for that particular pixel before acquiring
another pixel. If the images are acquired using a wide-field
imager, a lock-in technique can be applied on a pixel-by-pixel
basis after all images are acquired.
Method of Producing a Background-Free Image by Exploiting Excited
State Lifetime of FNDs
Referring now to FIG. 5, another imaging method 500 is
provided.
In step S502, an absorption wavelength (e.g., a brief pulse of 5 ns
or less) is applied to an object of interest impregnated with
fluorescent nanodiamonds. This absorption wavelength will excite
FNDs, but may also excite various background elements such as
endogenous proteins.
In step S504, after a delay of at least at least about 6
nanoseconds, acquiring a fluorescent image of the object of
interest. After 6 nanoseconds, most (if not all) background
fluorescence will have dissipated, while the FNDs continue to emit
photons. Thus, a background-free image can be captured without the
need for the image processing algorithms described above.
Computer Implementation of Methods
Referring now to FIG. 6, the methods described herein can be
implemented in hardware and/or software. For example, FIG. 6
depicts a system 600 including a fluorescent imaging device 602
such as a fluorescent microscope and a computer 604. The
fluorescent imaging device 602 can include an excitation light
source, an image sensor (e.g., including a charge-coupled device
(CCD), complementary metal-oxide-semiconductor (CMOS) chip,
photomultiplier tube (PMT), or avalanche photodiode (APD)), one or
more lenses, and a filter adapted to block undesired wavelengths.
The computer 604 can be a special-purpose or general-purpose
computer can be communicatively coupled with the fluorescent
imaging device 602 via communication standards such as parallel or
serial ports, Universal Serial Bus (USB), USB 2.0, Firewire,
Ethernet, Gigabit Ethernet, and the like.
As understood by those of skill in the art, computer 604 can
include various components such as a display device, a processor,
and/or a storage device.
Display device can be any device capable of displaying graphics
and/or text. Examples of display devices include a cathode ray tube
(CRT), a plasma display, a liquid crystal display (LCD), an organic
light-emitting diode display (OLED), a light-emitting diode (LED)
display, an electroluminescent display (ELD), a surface-conduction
electron-emitter display (SED), a field emission display (FED), a
nano-emissive display (NED), an electrophoretic display, a
bichromal ball display, an interferometric modulator display, a
bistable nematic liquid crystal display, and the like.
Processor is an electronic device (also known as a central
processing unit or microprocessor) capable of executing
instructions stored as hardware and/or software. Suitable
processors are available from manufacturers such as Intel
Corporation of Santa Clara, Calif. or Advanced Micro Devices (AMD)
of Sunnyvale, Calif.
Storage device can include persistent storage devices such as
magnetic media (e.g. tapes, disks), optical media (e.g. CD-ROM,
CD-R, CD-RW, DVD, HD DVD, BLU-RAY DISK.RTM., Laserdisk), punch
cards, and the like. Storage device can also include temporary
storage devices known as memory (e.g., random access memory).
Motion Correction of Movies
In some embodiments, motion correction is applied to compensate for
motion in the camera and/or the subject. For example, motion
correction can be applied to a time series of images to correct for
in-plane motion at the sub-pixel level.
In one embodiment, a non-rigid deformation map is calculated
pairwise between each image and a common reference image using an
optical flow method, which iteratively maximizes the local
cross-correlation image subsets at different resolutions. The
initial time frame can be chosen as a common reference for both
images series acquired with external field ON and OFF in order so
that the ON and OFF images are co-registered. A subpixel spline
based interpolation can be used for application of the non-rigid
deformation to minimize loss of spatial resolution. Following
motion correction, the time series of images can be averaged to
reduce random fluctuation due to noise thereby creating an average
image for the ON and OFF. A difference image between ON and OFF
motion corrected averages can be used to analyze the contrast of
the probe signal by subtraction of the background tissue. This
signal from the nanodiamond can then be overlaid on the starting
image.
Fluorescent Nanodiamonds and Compositions Thereof
As described herein, the present invention relates to use of
fluorescent nanodiamonds to image an object of interest.
Fluorescent nanodiamonds suitable for use in the present invention
and methods for making such fluorescent molecules are well-known in
the art. See, e.g., Nanodiamonds: Applications in Biology and
Nanoscale Medicine (D. Ho ed., Springer 2009); Molecular Imaging
(R. Weissleder et al. eds., 2010); Medical Nanotechnology and
Nanomedicine (Perspectives in Nanotechnology) (H. F. Tibbals ed.,
CRC Press 2010); Molecular Fluorescence (B. Valeur ed., Wiley-VCH
2012); Introduction to Nanomedicine and Nanobioengineering (Wiley
Series in Biomedical Engineering and Multi-Disciplinary Integrated
Systems) (P. N. Prasad ed., Wiley 2012); Yu et al., J. Am. Chem.
Soc. 127:17604-17605 (2005); Mochalin et al., Nature Nanotechnology
7:11-23 (2012); Epstein et al., Nature Physics 1:94-98 (2005); Chow
et al., Sci. Transl. Med. 3:73ra21 (2011); Awschalom et al., Sci.
Am. 297:84-91 (2007); and Wilson, Phys. Today 64:17-18 (2011).
The fluorescent nanodiamonds can be provided as a solution,
emulsion, suspension, microsphere, particle, microparticle,
nanoparticle, liposomes, and the like.
In aspects of the invention, the fluorescent nanodiamonds are
directly contacted with a sample (e.g., when impregnating the
fluorescent nanodiamond in a sample in vitro, ex vivo, in situ,
etc.). For example, fluorescent nanodiamonds can be impregnated in
a sample by injection (e.g., microinjection) or use of a delivery
vehicle. Suitable delivery vehicles are well-known in the art.
Nonlimiting examples include lipid vesicles or other polymer
carrier materials, lipoplexes (see, e.g., U.S. Patent Application
Publication No. 2003/0203865; and Zhang et al., J. Control Release,
100:165-180 (2004)), pH-sensitive lipoplexes (see, e.g., U.S.
Patent Application Publication No. 2002/0192275), reversibly-masked
lipoplexes (see, e.g., U.S. Patent Application Publication Nos.
2003/0180950), cationic lipid-based compositions (see, e.g., U.S.
Pat. No. 6,756,054; and U.S. Patent Application Publication No.
2005/0234232), cationic liposomes (see, e.g., U.S. Patent
Application Publication Nos. 2003/0229040, 2002/0160038, and
2002/0012998; U.S. Pat. No. 5,908,635; and International
Publication No. WO 01/72283), anionic liposomes (see, e.g., U.S.
Patent Application Publication No. 2003/0026831), pH-sensitive
liposomes (see, e.g., U.S. Patent Application Publication No.
2002/0192274; and Australian Publication No. 2003/210303),
antibody-coated liposomes (see, e.g., U.S. Patent Application
Publication No. 2003/0108597; and International Publication No. WO
00/50008), cell-type-specific liposomes (see, e.g., U.S. Patent
Application Publication No. 2003/0198664), liposomes containing
nucleic acid and peptides (see, e.g., U.S. Pat. No. 6,207,456),
liposomes containing lipids derivatized with releasable hydrophilic
polymers (see, e.g., U.S. Patent Application Publication No.
2003/0031704), lipid-entrapped molecules (see, e.g., International
Publication Nos. WO 03/057190 and WO 03/059322), lipid-encapsulated
molecules (see, e.g., U.S. Patent Application Publication No.
2003/0129221; and U.S. Pat. No. 5,756,122), other liposomal
compositions (see, e.g., U.S. Patent Application Publication Nos.
2003/0035829 and 2003/0072794; and U.S. Pat. No. 6,200,599),
stabilized mixtures of liposomes and emulsions (see, e.g., European
Publication No. EP 1 304 160), emulsion compositions (see, e.g.,
U.S. Pat. No. 6,747,014), and micro-emulsions (see, e.g., U.S.
Patent Application Publication No. 2005/0037086).
In some aspects of the invention, the fluorescent nanodiamonds are
administered in vivo to a subject (e.g., delivering the fluorescent
nanodiamond to a target object within the subject, including, but
not limited to, a target cell, tissue, organ, organism, infectious
agent, a virus, a bacteria, fungus, a parasite, and the like). When
administered to a subject, the fluorescent nanodiamonds can be
provided as a pharmaceutical composition including a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable" means approved by a regulatory agency or listed in the
U.S. Pharmacopeia or other generally recognized pharmacopeia for
use in animals, and more particularly in humans. The term "carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil, olive oil, gel (e.g.,
hydrogel), and the like. Saline is an exemplary carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable
solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol, and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the like. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. Examples of suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin, the contents of which are hereby incorporated by
reference in its entirety.
In embodiments, the imaging agent may be administered through
different routes, including, but not limited to, oral, parenteral,
buccal and sublingual, rectal, aerosol, nasal, intramuscular,
subcutaneous, intradermal, and topical. The term parenteral as used
herein includes, for example, intraocular, subcutaneous,
intraperitoneal, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrastemal,
intrathecal, intralesional, and intracranial injection, or other
infusion techniques. One of ordinary skill in the art would readily
understand that the formulation should suit the mode of
administration.
Formulations suitable for administration include aqueous and
non-aqueous sterile solutions, which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example, sealed ampoules
and vials, and may be stored in a freeze-dried (lyophilized)
condition requiring only the addition of the sterile liquid
carrier, for example, water, immediately prior to use.
Extemporaneous solutions and suspensions may be prepared from
sterile powders, granules and tablets commonly used by one of
ordinary skill in the art.
For oral administration in the form of a tablet or capsule, the
fluorescent nanodiamonds can be combined with an oral, non-toxic,
pharmaceutically acceptable, inert carrier such as lactose, starch,
sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium
phosphate, calcium sulfate, mannitol, sorbitol and the like. For
oral administration in liquid form, the fluorescent nanodiamonds
can be combined with any oral, non-toxic, pharmaceutically
acceptable inert carrier such as ethanol, glycerol, water, and the
like. Moreover, when desired or necessary, suitable binders,
lubricants, disintegrating agents, and coloring agents can also be
incorporated into the mixture. Suitable binders include starch,
gelatin, natural sugars such as glucose or .beta.-lactose, corn
sweeteners, natural and synthetic gums such as acacia, tragacanth,
or sodium alginate, carboxymethylcellulose, polyethylene glycol,
waxes, and the like. Lubricants used in these dosage forms include
sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride, and the like.
Disintegrators include, without limitation, starch, methyl
cellulose, agar, bentonite, xanthan gum, and the like.
The fluorescent nanodiamonds used in the methods of the present
invention can also be administered in the form of liposome delivery
systems. Such delivery systems are well known in the art, and
include but are not limited to, unilamellar vesicles, large
unilamallar vesicles, and multilamellar vesicles. Liposomes can be
formed from a variety of phospholipids, such as cholesterol,
stearylamine, or phosphatidylcholines.
The administration of the fluorescent nanodiamonds to a subject can
be by a general or local administration route. For example, the
fluorescent nanodiamonds may be administered to the subject such
that it is delivered throughout the body. Alternatively, the
fluorescent nanodiamonds can be administered to a specific organ or
tissue of interest.
The fluorescent nanodiamonds should have sufficient emission to
assure reliable diagnosis. The amount of fluorescent nanodiamonds
to be contacted with a sample or introduced into a subject in order
to provide for detection can readily be determined by those skilled
in the art. For example, increasing amounts of the fluorescent
nanodiamonds can be applied or given to a subject until the
fluorescent nanodiamonds are detected by the detection method of
choice. In addition, those skilled in the art are also familiar
with determining the amount of time sufficient for the fluorescent
nanodiamonds to become associated with a target object. The amount
of time necessary can easily be determined by introducing a
detectable amount of the fluorescent nanodiamonds into a subject
and then detecting the fluorescent nanodiamonds at various times
after administration.
In some aspects, the fluorescent nanodiamonds can be associated
with a molecule that preferentially binds to the target object.
Such molecules are well-known in the art, and include but are not
limited to target-binding agents having one or more target
recognition moieties for the selective binding of the
target-binding agents (and fluorescent nanodiamond) to a target
molecule. The target recognition moiety is configured to
specifically bind to a target molecule of a particular cell,
tissue, organ, receptor, surface antigen, organism/infectious
agent, and the like.
Examples of target recognition moieties include, but are not
limited to, an antigen, ligand, receptor, one member of a specific
binding pair, polyamide, peptide, carbohydrate, oligosaccharide,
polysaccharide, low density lipoprotein (LDL) or an apoprotein of
LDL, steroid, steroid derivative, hormone, hormone-mimic, lectin,
drug, antibiotic, aptamer, DNA, RNA, lipid, an antibody, an
antibody-related polypeptide, and the like. In embodiments, the
targeting moieties are polypeptides, carbohydrates, or lipids. The
targeting moieties can also be antibodies, antibody fragments, or
nanobodies. In other embodiments, the target recognition moiety can
be a molecule or a macromolecular structure (e.g., a liposome, a
micelle, a lipid vesicle, or the like) that preferentially
associates or binds to a particular tissue, receptor,
organism/infectious agent, and the like.
One of ordinary skill in the art would readily understand how to
make the fluorescent nanodiamond conjugates contemplated herein.
For example, the fluorescent nanodiamonds can be covalently or
non-covalently associated with the target binding agent/moiety. See
Vaijayanthimalal et al., Nanomedicine 4:47-55 (2009);
Vaijayanthimalal et al., Biomaterials 33: 7794-7802 (2012);
Hartmann et al., Chemistry--A European Journal 18:21, 6485-6492
(2012); Mochalin et al., Nat. Nanotechnology 7:11-23 (2012); Weng
et al., Diamond and Related Materials 22:96-104 (2012); Alhaddad et
al., Small 7:3087-3095 (2011); Krueger, J. Mater. Chem.
21:12571-12578 (2011); and Liu et al., Nanoscale Research Letters
5:1045-1050 (2010); Rurack, Supramolecular Chemistry Meets Hybrid
(Nano)Materials: A Brief Look Ahead, pages 689-700 of The
Supramolecular Chemistry of Organic-Inorganic Hybrid Materials
(Wiley, 2010).
In some embodiments, the FNDs are silica-coated FNDs. Methods for
creating silica-coated nanodiamonds are described in A. Bumb et
al., "Silica encapsulation of fluorescent nanodiamonds for
colloidal stability and facile surface functionalization," 135
Journal of the American Chemical Society 7815-18 (2013).
WORKING EXAMPLES
Working Example #1
Magnetic Modulation of FND Emission
FIG. 7A depicts the energy level diagram of NV.sup.- centers in
diamond showing spin-triplet (m.sub.s=0 and m.sub.s=.+-.1) ground
and excited states as well as the singlet metastable state.
NV.sup.- centers can be optically excited over a broad range of
wavelengths (450-650 nm) (green arrows). NV.sup.- centers in the
m.sub.s=.+-.1 sublevels of the excited states have a higher
probability to decay via the metastable state (grey dashed arrows)
than to the m.sub.s=.+-.1 sublevels of the ground state. From the
metastable state, NV.sup.- centers predominantly transition to the
m.sub.s=0 sublevel of the ground state without emitting visible
light. Therefore, in the absence of a magnetic field, NV.sup.-
centers are rapidly pumped into the m.sub.s=0 sublevel of the
ground state when excited. This results in an initial increase in
fluorescence emission intensity as steady state is reached. In the
presence of a magnetic field, the m.sub.s=0 and m.sub.s=.+-.1
states are mixed, making the decay pathway through the metastable
singlet state accessible and therefore decreasing the fluorescence
emission intensity.
FIG. 7A depicts a field of view containing FNDs. FIG. 7B depicts
the intensity modulation of the FND depicted in FIG. 7A upon
application of a modulating magnetic field with 0.1 Hz frequency
and 100 Gauss amplitude.
To show magnetic modulation of FND emission, a coverslip was
prepared with FNDs. 500 .mu.l of 1 mg/ml poly-L-lysine (PLL) in PBS
buffer was mixed with silica-coated FNDs, deposited on a #1
coverslip, and incubated overnight. A flow cell was made using
double-sided tape to attach the coverslip to a glass slide.
Movies were obtained using a CARL ZEISS.RTM. LSMS LIVE microscope.
Each frame of the movie was a scanning confocal image with 250 ms
time resolution. A 10.times. ZEISS.RTM. objective with 0.3 NA (EC
Plan-Neofluar) was used to introduce the excitation light to
stimulate the FNDs and to collect the FND emission. Samples were
excited at 532 nm, emission was filtered using a long pass filter
LP650 and detected using a photomultiplier tube (PMT).
An electromagnet (APW Company, Item #EM400-12-212, 4.0'' Diameter
Round Electromagnet) was powered by square wave voltage signal with
an amplitude of 0 or 12V and zero offset. The sample was placed
.about.13 mm away from the face of the magnet, where the magnetic
field strength was .about.100 Gauss.
Working Example #2
Background-Free Imaging by Pairwise Subtraction of Frames with and
Without Magnetic Field
FIG. 8A is an image of a field of view with .about.40 nm FNDs
containing .about.15 NV.sup.- imaged as in FIG. 7B. FIG. 8B is an
image of the same field of view after introducing .about.1 .mu.M
ALEXA FLUOR.RTM. 647 dye solution into the flow cell. This dye has
similar emission characteristics as the FNDs so the fluorescence
from the FNDs is masked by the background fluorescence of high
concentration of the ALEXA FLUOR.RTM. 647 dye. FIG. 8C is an image
of the same field of view after processing images in the presence
of the high ALEXA FLUOR.RTM. 647 dye background (as in FIG. 8B).
The difference between pairs of images collected with and without
the magnetic field was computed and 1,000 of these difference
images were averaged together to generate the processed image.
Through this processing, images of the diamonds shown in FIG. 8A
are recovered from images with high background like FIG. 8B.
Working Example #3
Background-Free Imaging Using Wide-Field Lock-In Detection
FIG. 9A shows a frame of a movie (1000 frames with time resolution
0.25 s) taken by a scanning confocal microscope. The details of the
imaging are identical to those in FIG. 7B above. A modulating
magnetic field with 0.1 Hz frequency and 100 Gauss amplitude was
applied during the acquisition of the movie.
FIG. 9B shows the wide field background-free image after processing
the movie pixel-by-pixel using a lock-in algorithm.
FIG. 9C depicts the application of the lock-in algorithm to a
bright pixel (horizontal coordinate, x=109 and vertical coordinate,
y=111 from the top left corner of the image in FIG. 9A)
corresponding to a FND. The top left panel of FIG. 9C illustrates
the pixel values as a function of time. The top right panel of FIG.
9C illustrates the fast Fourier transform (FFT) as a function of
frequency. The middle left panel of FIG. 9C depicts the pixel
values from the top left panel multiplied by a reference sine wave
1+sin(2.pi.*0.1*t). The middle right panel of FIG. 9C depicts the
corresponding FFT. The bottom left panel of FIG. 9C shows the pixel
values multiplied with a reference cosine wave 1+cos(2.pi.*0.1*t).
The bottom right panel of FIG. 9C depicts the corresponding
FFT.
FIG. 9D illustrates the same lock-in algorithm applied to a dark
pixel (horizontal coordinate, x=137 and vertical coordinate, y=107
from the top left corner of the image in FIG. 9A) corresponding to
the background.
Referring to both FIGS. 9C and 9D, vertical dashed lines at 0.2 Hz
are guides to indicate the values at twice the reference frequency.
Means of three points around 0.2 Hz in the sine and cosine FFTs
were calculated for each pixel. The background-free image in FIG.
9B corresponds to the sine and cosine means added in quadrature,
i.e, i=sqrt(i cos.sup.2+i sin.sup.2), where i is the mean intensity
and i cos and i sin correspond to the means calculated from the FFT
of the signal multiplied by the cosine and sine functions
respectively. Means of the pixel values over 1000 frames were 173
and 18 for the pixels at (109,111) and (137,107) before applying
the lock-in algorithm giving a signal-to-noise ratio of .about.10.
Means of the FFT amplitudes for three points around 0.2 Hz are
21.42 and 0.28 for the pixels at (109,111) and (137,107) after
applying the lock-in algorithm giving a signal-to-noise ratio of
.about.77. Thus the lock-in algorithm increased the signal to noise
ratio by a factor of .about.8. Computations were implemented in
MATLAB.RTM. software.
Working Example #4
Background-Free Imaging of Sentinal Lymph Node In Vivo Using Wide
Field Pairwise Image Subtraction and Lock-In Detection
FIG. 10A depicts an image of the forequarters of a mouse obtained
using conventional spectral unmixing methods to separate the
emission of the FNDs (top right inset, red in overlay) from
background fluorescence (top left inset, white in overlay). FNDs
were not detected through the skin in the draining axillary lymph
node. FIG. 10B depicts an image of the same mouse obtained by
averaging 475 pairwise-subtracted images with and without the
magnetic field. The processed image (top right inset, red in
overlay) was overlaid on an unprocessed image obtained with the
magnetic field off (top left inset, white in overlay). The white
arrows point to the injection site in the footpad and the location
of the auxiliary (sentinel) lymph node. Signal from the FNDs in the
lymph node is clearly detected. FIG. 10C depicts an image obtained
by lock-in detection of emission from FNDs from the same images
used to generate FIG. 10B using the algorithm described in FIG. 9
(top right inset, red in overlay) overlaid on the unprocessed image
obtained with the magnetic field off (top left inset, white in
overlay). FIG. 10D depicts an image of the same mouse's open chest
cavity by averaging of images obtained by pairwise subtracting
images with and without the magnetic field. The processed image
(bottom inset, red in overlay) was overlaid on an unprocessed image
obtained with the magnetic field off (top inset, white in overlay).
The white arrows point to the injection site in the front footpad
and the location of the auxiliary lymph node. FIG. 10E depicts an
image obtained from lock-in detection of emission from FNDs from
the same images used to generate FIG. 10D (bottom inset, red in
overlay) overlaid on the unprocessed image obtained with the
magnetic field off (top inset, white in overlay). In the partially
dissected mouse, localization of the FNDs to the lymph node can be
clearly seen. FIG. 10F depicts the pixel values as a function time
corresponding to the selected points in FIG. 10B and FIG. 10D. The
pixels selected were over (1) the axillary lymph node and (2) a
negative control on the skin in FIG. 10B and (3) the axillary lymph
node and (4) a negative control on a rib in FIG. 10D. Signal
modulation as a result of the applied magnetic field is clearly
visible in the lymph node through the skin, as well as when the
chest cavity was opened. Meanwhile, the skin and rib showed random
signal as would be expected for the negative control.
Female athymic (nu/nu) mice were purchased from Charles River
Laboratories at 4-6 weeks of age and housed in a specific
pathogen-free American Association for Laboratory Animal Care
approved facility. All experiments were approved by the National
Cancer Institute's Animal Care and Use Committee. Mice were
anesthetized using gas mixtures of 1.5-2.5% isoflurane in O.sub.2
to maintain a respiration rate of .about.30 bpm during the
injection procedure. A volume of 10 .mu.L of an .about.80 mg/mL
silica-coated nanodiamond (.about.100 nm) solution in PBS (pH7.4)
was intradermally injected into the front foot pad of each mouse.
Previous studies have shown that the primary draining LNs from this
injection site are the axillary and lateral thoracic LNs.
At 24 hours post-injection, the mice were sacrificed and optical
imaging was performed using a MAESTRO.TM. CRi spectroscopic optical
camera (excitation filter 523 nm, emission filter 675 nm long
pass). Images were also taken with magnetic modulation in a
UVP.RTM. BIOSPECTRUM.RTM. Imaging System equipped with a
BIOLITE.RTM. MultiSpectral Light Source (excitation filter 525 nm,
emission filter 650 nm long pass) with additional illumination
provided by a green laser (Z-Bolt.RTM. #DPSS-100) achieving
.about.1 mW/cm.sup.2 intensity. Each mouse was imaged in the
instrument on a non-magnetic stage under which the permanent magnet
(Catalog #DZ08-N52, K&J Magnetics) could be slid in and out.
Images were captured in series with magnetic fields off and on and
movies stitched together in ImageJ software available at
http://rsb.info.nih.gov/ij.
FIG. 11A is an image of a lymph node (LN) injected with
silica-coated FNDs without applied magnetic field. FIG. 11B is an
image of the same field of view during application of a magnetic
field of .about.100 Gauss. FIG. 11B was then subtracted from FIG.
11A. FIG. 11C depicts the sum of 20 such subtracted images. The
FNDs are observed as bright spots in the otherwise dark field.
FIGS. 11D and 11E depict the sums of 20 such subtractions when
magnetic field was always OFF and ON, respectively.
Female athymic (nu/nu) mice were purchased from Charles River
Laboratories of Wilmington, Mass. at 4-6 weeks of age and housed in
a specific pathogen-free American Association for Laboratory Animal
Care approved facility. Animals were used between 6 and 8 weeks of
age, and experiments were approved by the National Cancer
Institute's Animal Care and User Committee. Lymphadenectomy of the
axillary and lateral thoracic lymph nodes (LN) of the mice was
performed. The excised LN was injected with silica-coated FNDs. The
LN was then fixed in 10% formalin for 3 days, washed with PBS, and
mounted on a SUPERFROST PLUS.RTM. slide (obtained from VWR) using
PERMOUNT.RTM. medium (obtained from Fisher Scientific) to adhere
the coverslip.
Background-free images were obtained by using a Neodymium permanent
magnet (obtained from K&J Magnetics of Jamison, Pa.). A CARL
ZEISS.RTM. LSM5 LIVE microscope was used to obtain the movies. Each
frame of the movie was a scanning confocal image with 1 s time
resolution. A 10.times. ZEISS.RTM. objective with 0.3 NA (EC
Plan-Neofluar) was used to excite the FNDs and collect the FND
emission. Samples were excited at 532 nm. Emission was filtered
using a long pass filter (bandpass of 560-675 nm) and detected
using a PMT.
Working Example #5
Background-Free Imaging Using Fluorescence Lifetime Imaging
(FLIM)
FIG. 12A is a two-photon FLIM image of a different region of the
same LN used in FIGS. 11A-11E. Longer lifetimes (indicated by red
color) are believed to be due to FNDs, for which the reported
lifetimes range from .about.10-20 ns. FIG. 12B is a background-free
image of the same field of view obtained by subtracting images
without and with a magnetic field, and adding 10 such
subtractions.
A LEICA.RTM. TCS SP5 scanning confocal system was customized to
obtain both the fluorescent lifetime imaging microscopy (FLIM)
image (FIG. 12A) and the background-free confocal image in which a
Neodymium permanent magnet was used to apply the magnetic field
(.about.100 Gauss) (FIG. 12B). For FIG. 12B, 10 subtractions of
images with and without magnetic fields were added to obtain the
final image. An oil immersion objective (LEICA.RTM. HCX PL APO CS
40.0.times. 1.25 NA Oil UV) was used both to excite and to collect
the emission. For the FLIM image, the sample was excited at 930 nm
using a SPECTRA-PHYSICS.RTM. MAI TAI.RTM. laser. For FLIM imaging,
emission from the FNDs in the LN was filtered using a band pass
filter (BP 607-683 nm) and was detected using a PICOQUANT.TM.
PICOHARP.TM. 300 Time-Correlated Single Photon Counting (TCSPC)
system fitted with single photon avalanche diode (SPAD). For the
scanning confocal image (400 Hz scanning speed) of the same field
of view (FOV), the sample was excited at 561 nm and the emission
was filtered using band pass filter (600-789 nm) and was detected
using a PMT.
Conclusion
The present subject matter provides unique, elegant methods that
can be easily incorporated with existing microscope or animal
imaging systems without coming in contact with the sample. No
complicated and error-prone spectral unmixing is necessary.
Fluorescence of commonly-used stains and labels cannot be modulated
selectively using a magnetic field. Chemical or optical
modifications of commonly used labels such as GFP and dyes are
possible, but these modifications are difficult to implement, are
invasive, and can possibly induce unwanted changes.
EQUIVALENTS
While the present subject matter has been described with reference
to the above embodiments, it will be understood by those skilled in
the art that various changes can be made and equivalents can be
substituted for elements thereof without departing from the scope
of the subject matter. In addition, many modifications can be made
to adapt a particular situation or material to the teachings of the
subject matter without departing from the essential scope thereof.
Therefore, it is intended that the subject matter not be limited to
the particular embodiment disclosed as the best mode contemplated
for carrying out this subject matter, but that the subject matter
will include all embodiments falling within the scope of the
appended claims.
The functions of several elements may, in alternative embodiments,
be carried out by fewer elements, or a single element. Similarly,
in some embodiments, any functional element may perform fewer, or
different, operations than those described with respect to the
illustrated embodiment. Also, functional elements (e.g., modules,
databases, computers, and the like) shown as distinct for purposes
of illustration may be incorporated within other functional
elements, separated in different hardware or distributed in a
particular implementation.
While certain embodiments according to the present subject matter
have been described, the present subject matter is not limited to
just the described embodiments. Various changes and/or
modifications can be made to any of the described embodiments
without departing from the spirit or scope of the present subject
matter. Also, various combinations of elements, steps, features,
and/or aspects of the described embodiments are possible and
contemplated even if such combinations are not expressly identified
herein.
INCORPORATION BY REFERENCE
The entire contents of all patents, published patent applications,
and other references cited herein are hereby expressly incorporated
herein in their entireties by reference.
* * * * *
References